EAAT2 promoter and uses thereof

ABSTRACT

The present invention provides nucleic acid molecules comprising the EAAT2 promoter, as well as screening assays useful for identifying compounds which modulate the activity of the EAAT2 promoter, and methods of treating neurological disorders comprising administration of EAAT2 promoter modulators.

RELATED APPLICATIONS

This instant application is a 371 of PCT/US03/04414, filed Feb. 14,2003, which claims benefit of U.S. Provisional Application 60/357,179,filed Feb. 15, 2002, the entire contents of which are incorporatedherein by this reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention features the EAAT2 promoter and uses thereof. Inone aspect, the invention relates to novel nucleic acid moleculescomprising the EAAT2 promoter. In a related aspect, the inventionprovides methods for identifying, analyzing, and using thepolynucleotides. Further provided are screening methods for detectingtherapeutic compounds with capacity to treat neurological disorders.

2. Background

Neurological disorders can significantly impact the central nervoussystem (CNS) and motor neuron units. For example, certain neurologicaldisorders of the CNS are known to adversely affect the brain andassociated structures. Neurological disorders affecting motor neuronunits have been grouped into motor neuron diseases and peripheralneuropathies. See generally Kandel, E. R. et al; (1991) in Principles ofNeuroscience, Appleton & Lange, Norwalk, Conn.; and Rowland, L. P. (ed.)(1982) in Human Motor Neuron Diseases, New York, Raven Press.

An illustrative motor neuron disease is amyotrophic lateral sclerosis(ALS). ALS has been reported to be a chronic neuromuscular disorderhaving recognized clinical manifestations. For example, it has beensuggested that degeneration of cortical and spinal/bulbar motor neuronsmay play a key role in the disorder. ALS is nearly always fatal. About95% of all ALS cases are sporadic, with many of the remaining casesshowing autosomal dominant inheritance. See e.g., Kuncl R. W. et al.,(1992) Motor Neuron Diseases in Diseases of the Nervous System, Asburyet al. eds. (Philadelphia W. B. Saunders) pp. 1179-1208; Brown, R. H.,(1996) Amer. Neurol. 30:145; Siddique, T. and Deng., H. X. (1996) Hum.Mol. Genetics 5:1465).

Specific CNS disorders have been also described. In particular, somehave been attributed to cholinergic, dopaminergic, adrenergic,serotonergic deficiencies or combinations thereof. CNS disorders ofsevere impact include pre-senile dementia (sometimes referred to asAlzheimer's disease (AD) or early-onset Alzheimer's disease), seniledementia (dementia of the Alzheimer's type), Parkinson's disease (PD),and Huntington's disease (HD, sometimes referenced as Huntington'schorea). Such CNS disorders are well-represented in the humanpopulation. See generally; Gusella, J. F. et al. (1983) Nature 306: 234;Borlauer. W. and Jprmuloewoca. P. (eds.) (1976); Adv. in Parkinsonism:Biochemistry, Physiology, Treatment. Fifth International Symposium onParkinson's Disease (Vienna) Basel: Roche; and references cited therein.

Significant attention has been directed towards understanding theetiology of motor neuron diseases. For example, abnormal levels ofcertain excitotoxic neurotransmitters have been reported to adverselycontribute to many motor neuron diseases. In particular,glutamate-mediated excitotoxicity is recognized to have a critical rolein ALS. See e.g., Rothstein J. D. et al., (1990) Ann. Neurol. 28: 18.;Rothstein J. D. et al. (1992) N. Engl. Med. 326: 1464; Rothstein J. D.et al. (1993) PNAS (USA) 90: 6591; and Lacomblez, L. et al., (1996)Lancet 347: 1179.

There has been substantial efforts towards understanding mechanisms forreducing glutamate levels in the nervous system. For example,high-affinity, sodium-dependent glutamate transport is one reportedmeans of inactivating glutamate. In particular, astrocytic excitatoryamino acid transporter 2 (EAAT2) proteins are believed to havesubstantial functions in that inactivation. See e.g., Rothstein J. D. etal. (1994) Neuron 28: 18; Rothstein J. D. et al., (1995) Ann. Neurol.38: 78, and references cited therein.

In particular, investigations have suggested that EAAT2 is a predominantglutamate transporter. More particularly, certain antisense knockdownstudies have been reported to demonstrate that EAAT2 loss can lead toexcitotoxic neuronal degeneration and progressive motor impairment.Studies of ALS and other neurodegenerative disorders have relatedimpaired glutamate transport to loss of the EAAT2 protein. Inparticular, up to 60% to 70% of the sporadic ALS patients examined havea 30% to 95% loss of the EAAT2 protein. See e.g., Haugeto et al., supra;Rothstein J. D., et al., (1996) Neuron 16: 675; Bristol, L. A. andRothstein, J. D. (1996) Ann. Neurol. 39: 676.

There have been attempts to treat or prevent neurological disorders ofthe CNS and the motor neuron units. However, most existing therapies donot always stem the development or severity of the disorders inafflicted patients. See e.g., Rowell, (1987) Adv. Behav. Biol. 31: 191;Rinne, et al. Brain Res. (1991) 54: 167; U.S. Pat. No. 5,210,076 toBerliner; Yurek, D. M. (1990) Ann. Rev. Neurosci. 13: 415, and Rowlandet al. supra.

Accordingly, there is a need in the field for effective therapies fortreating neurological disorders.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofthe sequence of the EAAT2 promoter. Accordingly, the present inventionprovides nucleic acid molecules comprising the EAAT2 promoter, as wellas screening assays useful for identifying compounds which modulate theactivity of the EAAT2 promoter, and methods of treating neurologicaldisorders comprising administration of EAAT2 promoter modulators.

In one embodiment, the invention provides an isolated nucleic acidmolecule which comprises at least a portion of the EAAT2 promoter (e.g.,a P1 region, a P2 region, and/or a P3 region), or a complement thereof,wherein the nucleic acid molecule is capable of directing mRNAexpression from a promoterless reporter vector. In a preferredembodiment, the EAAT2 promoter comprises at least one SP1 binding site,an E-box motif, a GATA family transcription factor binding site, anNF-κB binding site, a WT1 binding site, a poly(dA:dT) region, apoly(dG:dT) region, and/or a cyclic AMP response element.

In a preferred embodiment, an isolated nucleic acid molecule comprisingthe EAAT2 promoter includes the nucleotide sequence set forth in SEQ IDNO:1, 2, 3, or 4, or a complement thereof. In another embodiment, anisolated nucleic acid molecule of the invention comprises a nucleotidesequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.25%, 99.5%,99.6%, 99.7%, 99.8%, or 99.9% identical to the nucleotide sequence ofSEQ ID NO:1, 2, 3, or 4, wherein the nucleic acid molecule is capable ofdirecting mRNA expression from a promoterless reporter vector, or acomplement thereof. In still another embodiment, an isolated nucleicacid molecule of the invention comprises at least 30 nucleotides of SEQID NO:1, 2, 3, or 4, wherein the nucleic acid molecule is capable ofdirecting mRNA expression from a promoterless reporter vector, or acomplement thereof.

In another embodiment, an isolated nucleic acid molecule of theinvention comprises an EAAT2 promoter or portion thereof and anoperatively linked cDNA molecule, for example, a reporter gene such asEAAT2, luciferase (e.g., firefly luciferase and Renilla luciferase),β-galactosidase, chloramphenicol acetyl transferase, or a fluorescentprotein (e.g., green fluorescent protein, enhanced green fluorescentprotein, red fluorescent protein, yellow fluorescent protein, enhancedyellow fluorescent protein, blue fluorescent protein, or cyanfluorescent protein).

In other embodiments, the invention provides vectors, includingexpression vectors and host cells comprising the nucleic acid moleculesof the invention, as well as methods of producing and/or detecting mRNAand polypeptides (e.g., reporter mRNA and polypeptides) encoded by DNAmolecules controlled by the EAAT2 promoter. The invention furherprovides methods of detecting the presence of the EAAT2 promoter.

In another embodiment, the invention provides methods of identifyingcompounds capable of modulating EAAT2 promoter activity to therebyidentify compounds capable of treating neurological disorders andpsychiatric disorders.

In still other embodiments, the invention provides methods of treating asubject having a neurological or psychiatric disorder comprisingadministering to the subject a therapeutically effective amount of anEAAT2 promoter modulator, thereby treating said subject having aneurological or psychiatric disorder.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depicts the nucleotide sequence of the EAAT2 promoter region(SEQ ID NO:1).

FIG. 2 depicts a schematic representation of the EAAT2 promoter regionshowing the identified promoter elements.

FIG. 3 depicts the results of a luciferase reporter activity assay usingthe P1, P2, and P3 EAAT2 promoter regions.

FIG. 4 depicts the BAC clone to be used in the generation of EAAT2promoter BAC transgenic mice.

FIG. 5 depicts the strategy to be used in the generation of EAAT2promoter BAC transgenic mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofthe sequence of the EAAT2 promoter. Accordingly, the present inventionprovides nucleic acid molecules comprising the EAAT2 promoter, as wellas screening assays useful for identifying compounds which modulate theactivity of the EAAT2 promoter, and methods of treating neurological andpsychiatric disorders comprising administration of EAAT2 promotermodulators.

The acidic amino acids glutamate (Glu) and aspartate are the predominantexcitatory neurotransmitters in the mammalian central nervous system(CNS). Although there are millimolar concentrations of these excitatoryamino acids (EAAs) in the brain, extracellular concentrations aremaintained in the low micromolar range to facilitate crisp synaptictransmission and to limit the neurotoxic potential of these EAAs. Afamily of Na⁺-dependent high affinity transporters is responsible forthe regulation and clearance of extracellular EAAs.

Glutamate and aspartate activate ligand-gated ion channels that arenamed for the agonists N-methyl-D-aspartate (NMDA),a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate.These ionotropic EAA receptors mediate rapid synaptic depolarization andare important for a number of other physiological processes, includingsynaptic plasticity and synapse development. The EAAs also activate afamily of metabotropic receptors coupled through G-proteins to secondmessenger systems or ion channels. It is well established that the EAAsare extremely important for normal brain function. However, there issubstantial evidence that an extracellular accumulation of EAAs andexcessive activation of EAA receptors also contributes to the neuronalcell death observed in acute insults to the CNS. The process known as,‘excitotoxicity’, may also contribute to neuronal loss observed inchronic neurodegenerative diseases, including amyotrophic lateralsclerosis (ALS).

The intracellular concentrations of glutamate (5-10 mM) and aspartate(1-5 mM) are 1000-fold to 10,000-fold greater than the extracellularconcentrations (<1-10 μM). Unlike many other neurotransmitters, there isno evidence that glutamate or aspartate is metabolized extracellularly.Instead, they are cleared from the extracellular space by transport intoneurons and astrocytes.

Several subtypes of Na⁺-dependent glutamate transporters have beenidentified through pharmacological strategies and cDNA cloning. Fiveknown distinct cDNA clones that express Na⁺-dependent high-affinityglutamate transport are referred to herein as GLT-1/EAAT2, EAAC1/EAAT3,GLAST/EAAT1, EAAT4, and EAAT5. There is also evidence for additionalheterogeneity of GLT-1 and GLAST that originates from alternate mRNAsplicing.

Expression of two of these transporters, GLT-1 and GLAST, is generallyrestricted to astroglia. Expression of two other transporters, EAAC1 andEAAT4, is generally restricted to neurons, and EAAT5 is thought to berestricted to retina Of the three transporters found in forebrain(GLT-1, GLAST, and EAAC1), GLT-1 appears to be the only transporter thatis specific to brain tissue, suggesting that GLT-1 expression iscontrolled by brain specific mechanisms.

Previously, it was thought that presynaptic transporters had a majorrole in the clearance of EAAs during synaptic transmission. This wasbased on the evidence that activity is enriched 2-fold in synaptosomalmembrane preparations compared to fractions enriched in mitochondria ormyelin. However, it is now known that these membrane preparationscontain resealed glial membranes and tremendous amounts of GLT-1protein. In addition, it has long been known that lesions of specificafferents result in a decrease in Na⁺-dependent transport in targetareas. For example, lesions of the cortical projections to the striatumresult in decreased uptake in striatal synaptosomes. These types ofstudies suggested that there was significant transport into presynapticterminals, but more recent studies have suggested that these lesionsreduce expression of the glial transporters.

Evidence from several complementary strategies strongly suggests thatGLT-1 mediates the bulk of Na⁺-dependent transport of EAAs in the CNS.For example, the pharmacological properties of GLT-1 parallel thepredominant component of activity observed in rat brain membranes. Basedon the enrichment required to purify GLT-1 to homogeneity, it is thoughtthat GLT-1 represents approximately 1% of total brain protein. Selectiveimmunoprecipitation of GLT-1 from solubilized forebrain tissue andreconstitution of the remaining protein in liposomes, suggests thatGLT-1 mediates 90% of transport activity. Anti-sense knock-down of GLT-1results in the dramatic reductions in synaptosomal transporter activityin several forebrain regions. Synaptosomal uptake in mice geneticallydeleted of GLT-1 is 5% of normal. Finally, electrophysiologicalrecording of transporter mediated currents in brain preparationsstrongly suggest that GLT-1 has a primary role for the clearance ofglutamate during synaptic transmission in several forebrain regions.

The expression of GLT-1/EAAT2 is dynamically regulated both in vivo andin vitro. Although GLT-1 is the predominant transporter in the adultCNS, expression is rather low early in development and increases duringsynaptogenesis in both rats and humans. As described above, lesions ofprojections to a particular target nucleus results in decreasedexpression of both glial transporters, GLT-1 and GLAST. These datasuggest that the presence of neurons induces and/or maintains expressionof the glial transporters.

Several different groups have demonstrated decreased expression of GLT-1and/or GLAST in animal models of acute insults to the CNS, includingstroke and traumatic brain injury. A loss in GLT-1 expression has beendemonstrated in patients with ALS. Furthermore, there is evidence ofdecreased expression of these transporters in humans with chronicneurodegenerative diseases, including Alzheimer's Disease, andHuntington's Disease. Loss of GLT-1 is also a feature of the fatal braintumor, glioblastoma multiforma.

Even though GLT-1 expression is extremely high in vivo, ‘normal’astrocytes maintained in culture express essentially no detectable mRNAor protein. Co-culturing astrocytes with neurons induces glialexpression of GLT-1, suggesting that neurons induce and/or maintainexpression of GLT-1 in vitro. This effect of neurons is, at least inpart, mediated by a soluble secreted molecule. Several small moleculesmimic this effect of neurons, including dbcAMP, epidermal growth factor,pituitary adenylate cyclase-activating peptide, and immunophilin. In allof these cases the increases in GLT-1 protein expression are accompaniedby an increase in GLT-1 mRNA and a change in the morphology of theastrocytes that many believe are reminiscent of differentiation.

The effects of dbcAMP are blocked by an inhibitor of protein kinase A.It has been shown that the increase in GLT-1 expression induced bydbcAMP, epidermal growth factor, or neuron conditioned medium are allblocked by an inhibitor of either phosphatidylinositol 3-kinase or aninhibitor of the transcription factor NF-B. Otherwise, little is knownabout the mechanisms that actually control GLT-1 expression. Thus, theidentification of the EAAT2 promoter provides a valuable tool tounderstand EAAT2 regulation and to develop assays to control itssynthesis.

As used herein, the term “EAAT2” refers to the human astroglialglutamate transporter 2 gene. See, e.g., U.S. Pat. No. 5,658,782 whichdiscloses the human EAAT2 cDNA sequence, the disclosure of the which isspecifically incorporated herein by reference. As used herein, the term“GLT-1” refers to the rodent astroglial glutamate transporter 2 gene.

As used herein, the term “promoter” generally refers a region of genomicDNA, usually found 5′ to an mRNA transcription start site. Promoters areinvolved in regulating the timing and level of mRNA transcription andcontain, for example, binding sites for cellular proteins such as RNApolymerase and other transcription factors. As used interchangeablyherein, the terms “EAAT2 promoter”, “EAAT2 promoter region” and the likeinclude the region of genomic DNA found 5′ to the EAAT2 mRNAtranscription start site. In preferred embodiments, the EAAT2 promotercomprises SEQ ID NO:1, 2, 3, or 4, or fragments thereof. When insertedinto a promoterless reporter construct, preferred EAAT2 promoterfragments are able to direct transcription of the reporter gene.

In one embodiment, the EAAT2 promoter includes SEQ ID NO:1 (e.g.,nucleotides 1-4696 of SEQ ED NO:1). In another embodiment the EAAT2promoter includes a P1 region, which comprises nucleotides 733-3450 ofSEQ ID NO:1 (also set forth as SEQ ID NO:2). In another embodiment, theEAAT2 promoter includes a P2 region, which comprises nucleotides733-3186 of SEQ ID NO:1 (also set forth as SEQ ID NO:3). In stillanother embodiment, the EAAT2 promoter includes a P3 region, whichcomprises nucleotides 2590-3450 of SEQ ID NO:1 (also set forth as SEQ IDNO:4).

The EAAT2 promoter molecules of the present invention provide noveldiagnostic targets and therapeutic agents for neurological andpsychiatric disorders. As used herein, the term ‘neurological disorder’includes a disorder, disease or condition which affects the nervoussystem, e.g., the central nervous system. The neurological disordersthat can be treated in accord with the present invention includespecific disorders that have been reported to be associated withexcitotoxicity. Particularly included are specified neurologicaldisorders affecting motor neuron function. Neurological disordersinclude, but are not limited to, amyotrophic lateral sclerosis (ALS),trinucleotide repeat expansion disorders (e.g., Huntington's disease(HD), spinal and bulbar muscular atrophy, spinocerebellar ataxia types1, 2, 6, and 7, dentatorubropallidoluysian atrophy, and Machado-Josephdisease), α-synucleinopathies (e.g., Parkinson's disease (PD), dementiawith Lewy bodies (DLB), and multiple system atrophy (MSA)), multiplesclerosis (MS), Alzheimer's disease, brain tumors (e.g., glioblastoma),stroke/ischemia, cerebrovascular disease, epilepsy (e.g., temporal lobeepilepsy), HIV-associated dementia, Korsakoff's disease, pain, headaches(e.g., migraine headaches), Pick's disease, progressive supranuclearpalsy, Creutzfeldt-Jakob disease, Bell's Palsy, aphasia, sleepdisorders, glaucoma, and Meniere's disease.

As used herein, the term ‘psychiatric disorder’ refers diseases anddisorders of the mind, and includes diseases and disorders listed in theDiagnostic and Statistical Manual of Mental Disorders—Fourth Edition(DSM-IV), published by the American Psychiatric Association, WashingtonD.C. (1994). Psychiatric disorders include, but are not limited to,anxiety disorders (e.g., acute stress disorder agoraphobia, generalizedanxiety disorder, obsessive-compulsive disorder, panic disorder,posttraumatic stress disorder, separation anxiety disorder, socialphobia, and specific phobia), childhood disorders, (e.g.,attention-deficit/hyperactivity disorder, conduct disorder, andoppositional defiant disorder), eating disorders (e.g., anorexia nervosaand bulimia nervosa), mood disorders (e.g., depression, bipolardisorder, cyclothymic disorder, dysthymic disorder, and major depressivedisorder), personality disorders (e.g., antisocial personality disorder,avoidant personality disorder, borderline personality disorder,dependent personality disorder, histrionic personality disorder,narcissistic personality disorder, obsessive-compulsive personalitydisorder, paranoid personality disorder, schizoid personality disorder,and schizotypal personality disorder), psychotic disorders (e.g., briefpsychotic disorder, delusional disorder, schizoaffective disorder,schizophreniform disorder, schizophrenia, and shared psychoticdisorder), substance-related disorders (e.g., alcohol dependence,amphetamine dependence, cannabis dependence, cocaine dependence,hallucinogen dependence, inhalant dependence, nicotine dependence,opioid dependence, phencyclidine dependence, and sedative dependence),adjustment disorder, autism, delirium, dementia, multi-infarct dementia,learning and memory disorders (e.g., amnesia and age-related memoryloss), and Tourette's disorder.

As noted, neurological and psychiatric disorders of specific interestinclude those associated with abnormal release or removal of excitotoxicamino acids such as glutamate. Several CNS neuron types are especiallyadversely affected by excitotoxic glutamate. See e.g., Choi, D. W.(1988) Neuron 1: 623; and references cited therein. Specificallypreferred neurological disorders include AD, HD, PD with ALS beingespecially preferred.

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat comprise the EAAT2 promoter or biologically active portionsthereof, as well as nucleic acid fragments sufficient for use ashybridization probes to identify EAAT2 promoter containing nucleic acidmolecules and fragments for use as PCR primers for the amplification ormutation of EAAT2 promoter nucleic acid molecules. As used herein, theterm ‘nucleic acid molecule’ is intended generally to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

In general, optimal practice of the present invention can be achieved byuse of recognized manipulations. For example, techniques for isolatingmRNA, methods for making and screening cDNA libraries, purifying andanalyzing nucleic acids, methods for making recombinant vector DNA,cleaving DNA with restriction enzymes, ligating DNA, introducing DNAinto host cells by stable or transient means, culturing the host cells,methods for isolating and purifying polypeptides and making antibodiesare generally known in the field. See generally Sambrook et al.,Molecular Cloning (2d ed. 1989), and Ausubel et al., Current Protocolsin Molecular Biology, (1989) John Wiley & Sons, New York.

The term ‘isolated nucleic acid molecule’ includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term ‘isolated’ includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an ‘isolated’ nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated EAAT2 promoter nucleicacid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb or 0.1 kb of nucleotide sequences which naturally flank thenucleic acid molecule in genomic DNA of the cell from which the nucleicacid is derived. Moreover, an ‘isolated’ nucleic acid molecule can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, 2, 3, or 4, or aportion thereof, can be isolated using standard molecular biologytechniques and the sequence information provided herein. Using all or aportion of the nucleic acid sequence of SEQ ID NO:1, 2, 3, or 4 ashybridization probes, EAAT2 promoter nucleic acid molecules can beisolated using standard hybridization and cloning techniques (e.g., asdescribed in Sambrook, J. et al., supra).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1, 2, 3, or 4 can be isolated by the polymerase chain reaction(PCR) using synthetic oligonucleotide primers designed based upon thesequence of SEQ ID NO:1, 2, 3, or4.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to EAAT2 promoter nucleotidesequences can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In one embodiment, an isolated nucleic acid molecule of the inventioncomprises the nucleotide sequence shown in SEQ ID NO:1 (FIGS. 1A-1B).This DNA molecule comprises sequences encoding the human EAAT2 promoter.An isolated nucleic acid molecule of the invention may also comprisenucleotides 733-3450 of SEQ ID NO:1 (also set forth as SEQ ID NO:2).This DNA sequence comprises the P1 region of the EAAT2 promoter. Inanother embodiment, an isolated nucleic acid molecule of the inventioncomprises nucleotides 733-3186 of SEQ ID NO:1 (also set forth as SEQ IDNO:3). This DNA molecule comprises the P2 region of the EAAT2 promoter.In still another embodiment, an isolated nucleic acid molecule of theinvention comprises nucleotides 2590-3450 of SEQ ID NO:1 (also set forthas SEQ ID NO:4). This DNA molecule comprises the P3 region of the EAAT2promoter.

In still another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:1, 2, 3, or 4, or a portion ofany of these nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:1, 2, 3, or4 is one which is sufficiently complementary to the nucleotide sequenceshown in SEQ ID NO:1, 2, 3, or 4 such that it can hybridize to thenucleotide sequence shown in SEQ ID NO:1, 2, 3, or 4, thereby forming astable duplex. The term ‘complementary’ or like term refers to thehybridization or base pairing between nucleotides or nucleic acids, suchas, for instance, between the two strands of a double stranded DNAmolecule or between an oligonucleotide primer and a primer binding siteon a single stranded nucleic acid to be sequenced or amplified.Complementary nucleotides are, generally, A and T (or A and U), or C andG. Two single stranded RNA or DNA molecules are said to be substantiallycomplementary when the nucleotides of one strand, optimally aligned andcompared and with appropriate nucleotide insertions or deletions, pairwith at least about 95% of the nucleotides of the other strand, usuallyat least about 98%, and more preferably from about 99 to about 100%.Complementary polynucleotide sequences can be identified by a variety ofapproaches including use of well-known computer algorithms and software.

In still another embodiment, an isolated nucleic acid molecule of thepresent invention comprises a nucleotide sequence which is at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, 99.9% or more identical to the nucleotide sequence shown in SEQID NO:1, 2, 3, or 4 (e.g., to the entire length of the nucleotidesequence), or a portion or complement of any of these nucleotidesequences. In one embodiment, a nucleic acid molecule of the presentinvention comprises a nucleotide sequence which comprises part or all ofSEQ ID NO:1, 2, 3, or 4, or a complement thereof, and which is at least(or no greater than) 25, 30, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1250, 1300, 1350, 1400, 1450, 1500,1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 1994, 2000, 2050,2073, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600,2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200,3250, 3300, 3350, 3400, 3441, 3450, 3500, 3550, 3600, 3650, 3700, 3750,3800, 3841, 3850, 3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300,4350, 4400, 4450, 4500, 4550, 4600, 4650 or more nucleotides (e.g.,contiguous nucleotides) in length.

To determine the percent identity of two nucleic acid or amino acidsequences, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to a nucleotide sequencehaving 100 nucleotides, at least 30, preferably at least 40, morepreferably at least 50, even more preferably at least 60, and even morepreferably at least 70, 80, or 90 nucleotides are aligned). The aminoacid residues or nucleotides at corresponding amnino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available at onlinethrough the Genetics Computer Group), using either a Blossum 62 matrixor a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent identity between two nucleotide sequences isdetermined using the GAP program in the GCG software package (availableat online through the Genetics Computer Group), using a NWSgapdna.CMPmatrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of1, 2, 3, 4, 5, or 6. A preferred, non-limiting example of parameters tobe used in conjunction with the GAP program include a Blosum 62 scoringmatrix with a gap penalty of 12, a gap extend penalty of 4, and aframeshift gap penalty of 5.

In another embodiment, the percent identity between two amino acid ornucleotide sequences is determined using the algorithm of Meyers andMiller (Comput. Appl. Biosci. 4:11-17 (1988)) which has beenincorporated into the ALIGN program (version 2.0 or version 2.0U), usinga PAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to EAAT2 nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to EAAT2 proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See the website for the NationalCenter for Biotechnology Information.

The nucleic acid molecule of the invention can comprise only a portionof the nucleic acid sequence of SEQ ID NO:1, 2, 3, or 4, for example, afragment which can be used as a probe or primer or a fragment encoding aportion of an EAAT2 promoter. The probe/primer (e.g., oligonucleotide)typically comprises substantially purified oligonucleotide. Theoligonucleotide typically comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12 or 15,preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55,60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ IDNO:1, 2, 3, or 4, or a complement thereof.

Exemplary probes or primers are at least (or no greater than) 12 or 15,20 or 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more nucleotides inlength and/or comprise consecutive nucleotides of an isolated nucleicacid molecule described herein. Also included within the scope of thepresent invention are probes or primers comprising contiguous orconsecutive nucleotides of an isolated nucleic acid molecule describedherein, but for the difference of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 baseswithin the probe or primer sequence. Probes based on the EAAT2 promoternucleotide sequences can be used to detect (e.g., specifically detect)genomic sequences. In preferred embodiments, the probe further comprisesa label group attached thereto, e.g., the label group can be aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.In another embodiment a set of primers is provided, e.g., primerssuitable for use in a PCR, which can be used to amplify a selectedregion of an EAAT2 promoter sequence, e.g., a domain, region, site orother sequence described herein. The primers should be at least 5, 10,or 50 base pairs in length and less than 100, or less than 200, basepairs in length. The primers should be identical, or differ by nogreater than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases when compared to asequence disclosed herein or to the sequence of a naturally occurringvariant. Such probes can be used as a part of a diagnostic test kit foridentifying cells or tissue which misexpress an EAAT2 protein, such asby measuring a level of an EAAT2 promoter activity in a sample of cellsfrom a subject, e.g., determining whether a genomic EAAT2 promoter hasbeen mutated or deleted.

A nucleic acid fragment encoding a portion of an EAAT2 promoter can beprepared by isolating a portion of the nucleotide sequence of SEQ IDNO:1, 2, 3, or 4, inserting the portion of the EAAT2 promoter (e.g., bystandard recombinant methods) into a promoterless reporter vector (e.g.,a promoterless luciferase reporter vector such as pGL3, available fromPromega, Madison, Wis., and assessing the activity of the portion of theEAAT2 promoter to induce luciferase expression (e.g., when transientlytransfected into a cell). In an exemplary embodiment, the nucleic acidmolecule is at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,1200, 1250, 1300, 1350, 1400, 1250, 1300, 1350, 1400, 1450, 1500, 1550,1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 1994, 2000, 2050, 2073,2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650,2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250,3300, 3350, 3400, 3441, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800,3841, 3850, 3900, 3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350,4400, 4450, 4500, 4550, 4600, 4650 or more nucleotides in length.

In another embodiment, nucleic acid molecules of the invention cancomprise variants of the sequences disclosed herein. Nucleic acidvariants can be naturally occurring, such as allelic variants (samelocus), homologues (different locus), and orthologues (differentorganism, e.g., mouse) or can be non-naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.

Allelic variants result, for example, from DNA sequence polymorphismswithin a population (e.g., the human population). Such geneticpolymorphism in the EAAT2 promoter may exist among individuals within apopulation due to natural allelic variation.

Allelic variants of EAAT2 promoter include both functional andnon-functional EAAT2 promoters. Functional allelic variants arenaturally occurring nucleotide sequence variants of the EAAT2 promoterthat maintain the ability to, e.g., drive transcription of the EAAT2mRNA. Non-functional allelic variants are naturally occurring nucleotidesequence variants of the EAAT2 promoter that do not have the ability to,e.g., drive transcription of the EAAT2 mRNA, ATP, or that induce EAAT2transcription at levels higher than normally observed.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the EAAT2 promoters of the invention can be isolated basedon their homology to the EAAT2 promoter nucleic acids disclosed hereinusing the nucleic acid sequences disclosed herein, or a portionsthereof, as hybridization probes according to standard hybridizationtechniques under stringent hybridization conditions. Nucleic acidmolecules corresponding to natural allelic variants and homologues ofthe EAAT2 promoters of the invention can further be isolated by mappingto the same chromosome or locus as the EAAT2 gene.

Orthologues, homologues, and allelic variants can be identified usingmethods known in the art (e.g., by hybridization to an isolated nucleicacid molecule of the present invention, for example, under stringenthybridization conditions). In one embodiment, an isolated nucleic acidmolecule of the invention is at least 15, 20, 25, 30 or more nucleotidesin length and hybridizes under stringent conditions to the nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1, 2, 3, or 4.In other embodiments, the nucleic acid is at least 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1250, 1300,1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900,1950, 1994, 2000, 2050, 2073, 2100, 2150, 2200, 2250, 2300, 2350, 2400,2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000,3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3441, 3450, 3500, 3550,3600, 3650, 3700, 3750, 3800, 3841, 3850, 3900, 3950, 4000, 4050, 4100,4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650, ormore nucleotides in length.

As used herein, the term ‘hybridizes under stringent conditions’ isintended to describe conditions for hybridization and washing underwhich nucleotide sequences that are significantly identical orhomologous to each other remain hybridized to each other. Preferably,the conditions are such that sequences at least about 70%, morepreferably at least about 80%, even more preferably at least about 85%or 90% identical to each other remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, Inc. (1995), sections 2, 4, and 6. Additionalstringent conditions can be found in Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989), chapters 7, 9, and 11. A preferred, non-limiting example ofstringent hybridization conditions includes hybridization in 4× sodiumchloride/sodium citrate (SSC), at about 65-70° C. (or alternativelyhybridization in 4×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 1×SSC, at about 65-70° C. A preferred,non-limiting example of highly stringent hybridization conditionsincludes hybridization in 1×SSC, at about 65-70° C. (or alternativelyhybridization in 1×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 0.3×SSC, at about 65-70° C. A preferred,non-limiting example of reduced stringency hybridization conditionsincludes hybridization in 4×SSC at about 50-60° C. (or alternativelyhybridization in 6×SSC plus 50% formamide at about 40-45° C.) followedby one or more washes in 2×SSC, at about 50-60° C. Ranges intermediateto the above-recited values, e.g., at 65-70° C. or at 42-50° C. are alsointended to be encompassed by the present invention. SSPE (1×SSPE is0.15M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can be substitutedfor SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) in thehybridization and wash buffers; washes are performed for 15 minutes eachafter hybridization is complete. The hybridization temperature forhybrids anticipated to be less than 50 base pairs in length should be5-10° C. less than the melting temperature (T_(m)) of the hybrid, whereT_(m) is determined according to the following equations. For hybridsless than 18 base pairs in length, T_(m)(° C.)=2(# of A+T bases)+4(# ofG+C bases). For hybrids between 18 and 49 base pairs in length, T_(m)(°C.)=81.5+16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including but not limited to blockingagents (e.g., BSA or salmon or herring sperm carrier DNA), detergents(e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.When using nylon membranes, in particular, an additional preferred,non-limiting example of stringent hybridization conditions ishybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed byone or more washes at 0.02M NaH₂PO₄, 1% SDS at 65° C. (see e.g., Churchand Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995), oralternatively 0.2×SSC, 1% SDS.

Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO:1, 2,3, or 4 corresponds to a naturally-occurring nucleic acid molecule. Asused herein, a ‘naturally-occurring’ nucleic acid molecule refers to anRNA or DNA molecule having a nucleotide sequence that occurs in nature.

In addition to naturally-occurring allelic variants of the EAAT2promoter sequences that may exist in the population, the skilled artisanwill further appreciate that changes can be introduced by mutation intothe nucleotide sequences of SEQ ID NO:1, 2, 3, or 4, without alteringthe functional ability of the EAAT2 promoter sequences. In oneembodiment, the isolated nucleic acid molecule comprises a nucleotidesequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ IDNO:1, 2, 3, or 4, e.g., to the entire length of SEQ ID NO:1, 2, 3, or 4.

II. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, for examplerecombinant expression vectors, containing an EAAT2 promoter nucleicacid molecule. As used herein, the term ‘vector’ refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a ‘plasmid’, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as ‘expression vectors’. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, ‘plasmid’ and‘vector’ can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, ‘operably linked’ is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term ‘regulatory sequence’ isintended to include promoters, enhancers and other expression controlelements (e.g. polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel (1990) Methods Enzymol. 185:3-7.Regulatory sequences include those which direct constitutive expressionof a nucleotide sequence in many types of host cells and those whichdirect expression of the nucleotide sequence only in certain host cells(e.g., tissue-specific regulatory sequences). In a preferred embodiment,the regulatory sequences in the expression vectors of the invention arederived from the EAAT2 promoter of the invention, and the nucleic acidsequence to be expressed is a reporter gene, as described elsewhereherein. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, and the like. The expression vectors of the invention can beintroduced into host cells to thereby produce proteins or peptides,including fusion proteins or peptides, encoded by nucleic acids asdescribed herein.

In a preferred embodiment, a recombinant vector of the invention is apromoterless reporter vector. As used herein, a “promoterless reportervector” refers to a vector, preferably a plasmid, that contains areporter gene, but no promoter region capable of driving expression ofthe reporter gene mRNA. Instead of a promoter, the promoterless reportervector contains at least one site that can be cleaved by a restrictionenzyme, and into which can be inserted a DNA fragment of interest (usingstandard recombinant DNA methods). The vector can then be tested (e.g.,in an in vitro assay or a transient transfection assay) for the abilityof the inserted DNA fragment to act as a promoter to drive expression ofthe reporter gene. Examples of promoterless reporter vectors include,but are not limited to, the pGL3 (Promega, Madison, Wis.), pBBR RESO(MiBiTec, Gottingen, Germany), pAM990, pAM1414, pRL-null Vector(Promega), phRG-B, pDsRed-Express-1 (BD Biosciences/Clontech, Palo Alto,Calif.), pDsRed2-1 (BD Biosciences/Clontech), pECFP-1 (BDBiosciences/Clontech), pEGFP-1 (BD Biosciences/Clontech), pEYFP-1 (BDBiosciences/Clontech), pSVOATCAT (see Lok S. et al. (1989) Nucleic AcidsRes. 17:3563-82), pBLCAT5, pXP2, and pPD96.04. Many of these vectors areavailable commercially.

As used herein a “reporter” or a “reporter gene” refers to a nucleicacid molecule encoding a detectable marker. Preferred reporter genesinclude luciferase (e.g., firefly luciferase or Renilla luciferase),β-galactosidase, chloramphenicol acetyl transferase (CAT), and afluorescent protein (e.g., green fluorescent protein, red fluorescentprotein, yellow fluorescent protein, blue fluorescent protein, cyanfluorescent protein, or variants thereof, including enhanced variants).In another preferred embodiment, a preferred reporter gene is the EAAT2gene. Reporter genes must be detectable by a reporter assay. Reporterassays can measure the level of reporter gene expression or activity byany number of means, including measuring the level of reporter mRNA, thelevel of reporter protein, or the amount of reporter protein activity.

Methods for measuring mRNA levels are well-known in the art and include,but are not limited to, Northern blotting, RT-PCR, primer extension, andnuclease protection assays. Methods for measuring reporter proteinlevels are also well-known in the art and include, but are not limitedto, Western blotting, ELISA, and RIA assays. Reporter activity assaysare still further well-known in the art, and include luciferase assays,β-galactosidase, and chloramphenicol acetyl transferase (CAT) assays.Fluorescent protein activity can be measured by detecting fluorescence.EAAT2 (i.e., GLT-1) activity can be measured using a standard glutamatetransport assays (described elsewhere herein).

Accordingly, in one embodiment, the invention provides a method forproducing mRNA and/or protein molecules (e.g., reporter gene mRNA and/orprotein molecules), by culturing in a suitable medium a host cell of theinvention (e.g., a mammalian host cell such as a non-human mammaliancell) containing a recombinant expression vector containing the EAAT2promoter or a fragment thereof, such that the mRNA and/or protein isproduced.

The recombinant expression vectors of the invention are preferablydesigned for expression in eukaryotic cells (e.g., mammalian cells).Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro.

Another aspect of the invention pertains to host cells into which anEAAT2 promoter nucleic acid molecule of the invention is introduced,e.g., an EAAT2 promoter nucleic acid molecule within a vector (e.g., arecombinant expression vector) or an EAAT2 promoter nucleic acidmolecule containing sequences which allow it to homologously recombineinto a specific site of the host cell's genome. The terms ‘host cell’and ‘recombinant host cell’ are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, avector containing an EAAT2 promoter can be propagated and/or expressedin bacterial cells such as E. coli, insect cells, yeast or mammaliancells (such as Chinese hamster ovary cells (CHO), COS cells (e.g., COS7cells), C6 glioma cells, HEK 293T cells, or neurons). Other suitablehost cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms ‘transformation’ and ‘transfection’ are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify an d select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding an EAAT2 promoter or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) an mRNA orprotein (e.g., an EAAT2 mRNA or protein, or a reporter mRNA or protein)encoded by the nucleic acid molecule operatively linked to the EAAT2promoter. Accordingly, the invention further provides methods forproducing an mRNA or protein using the host cells of the invention. Inone embodiment, the method comprises culturing the host cell of theinvention (into which a recombinant expression vector containing theEAAT2 promoter and an operatively linked nucleic acid molecule (e.g., acDNA molecule) has been introduced) in a suitable medium such that mRNAand/or protein encoded by the operatively linked nucleic acid moleculeis produced. In another embodiment, the method further comprisesisolating the mRNA and/or protein from the medium or the host cell.

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichEAAT2 promoter sequences have been introduced. Such host cells can thenbe used to create non-human transgenic animals in which exogenous EAAT2promoter sequences have been introduced into their genome or homologousrecombinant animals in which endogenous EAAT2 promoter sequences havebeen altered. Such animals are useful for studying the function and/oractivity of an EAAT2 promoter and for identifying and/or evaluatingmodulators of EAAT2 promoter activity. As used herein, a ‘transgenicanimal’ is a non-human animal, preferably a mammal, more preferably arodent such as a rat or mouse, in which one or more of the cells of theanimal includes a transgene. Other examples of transgenic animalsinclude non-human primates, sheep, dogs, cows, goats, chickens,amphibians, and the like. A transgene is exogenous DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, a ‘homologousrecombinant animal’ is a non-human animal, preferably a mammal, morepreferably a mouse, in which an endogenous EAAT2 promoter has beenaltered by homologous recombination between the endogenous gene and anexogenous DNA molecule introduced into a cell of the animal, e.g., anembryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing anEAAT2 promoter-encoding nucleic acid into the male pronuclei of afertilized oocyte, e.g., by microinjection or retroviral infection, andallowing the oocyte to develop in a pseudopregnant female foster animal.The EAAT2 promoter cDNA sequence of SEQ ID NO:1, 2, 3, or 4, can beintroduced as a transgene into the genome of a non-human animal.Alternatively, a non-human homologue of a human EAAT2 promoter, such asa rat or mouse EAAT2 promoter, can be used as a transgene. Methods forgenerating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of an EAAT2 promoter transgene in its genome and/or expressionof a reporter gene operatively linked to the EAAT2 promoter transgene intissues or cells of the animals. A transgenic founder animal can then beused to breed additional animals carrying the transgene. Moreover,transgenic animals carrying a transgene containing an EAAT2 promoter canfurther be bred to other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of an EAAT2 promoter into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the EAAT2 promoter. The EAAT2 promoter can be ahuman gene (e.g., the cDNA of SEQ ID NO:1, 2, 3, or 4), but morepreferably, is a non-human homologue of a human EAAT2 promoter. Forexample, a mouse EAAT2 promoter gene can be used to construct ahomologous recombination nucleic acid molecule, e.g., a vector, suitablefor altering an endogenous EAAT2 promoter gene in the mouse genome. In apreferred embodiment, the homologous recombination nucleic acid moleculeis designed such that, upon homologous recombination, the endogenousEAAT2 promoter is functionally disrupted (i.e., no longer encodes afunctional protein; also referred to as a ‘knock out’ vector).Alternatively, the homologous recombination nucleic acid molecule can bedesigned such that, upon homologous recombination, the endogenous EAAT2promoter is mutated or otherwise altered. In the homologousrecombination nucleic acid molecule, the altered portion of the EAAT2promoter is flanked at its 5′ and 3′ ends by additional nucleic acidsequence from the region of the EAAT2 promoter to allow for homologousrecombination to occur between the exogenous EAAT2 promoter carried bythe homologous recombination nucleic acid molecule and an endogenousEAAT2 promoter in a cell, e.g., an embryonic stem cell. The additionalflanking EAAT2 promoter nucleic acid sequence is of sufficient lengthfor successful homologous recombination with the endogenous gene.Typically, several kilobases of flanking DNA (both at the 5′ and 3′ends) are included in the homologous recombination nucleic acid molecule(see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for adescription of homologous recombination vectors). The homologousrecombination nucleic acid molecule is introduced into a cell, e.g., anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced EAAT2 promoter has homologously recombined with theendogenous EAAT2 promoter are selected (see e.g., Li, E. et al. (1992)Cell 69:915). The selected cells can then be injected into a blastocystof an animal (e.g., a mouse) to form aggregation chimeras (see e.g.,Bradley, A., in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, Robertson, E. J. ed. (IRL, Oxford, 1987) pp. 113-152). Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Progeny harboringthe homologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination nucleic acid molecules, e.g.,vectors, or homologous recombinant animals are described further inBradley, A. (1991) Curr. Opin. Biotechnol. 2:823-829 and in PCTInternational Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO93/04169 by Berns et al.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(O) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyte and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g., the somatic cell, isisolated.

Transgenic and homologous recombinant animals of the invention can alsobe used to produce stable cell lines containing the EAAT2 promoter. Suchcell lines are useful because they can be made so that they do notoverexpress the transgene (as may happen in transient transfection), andtherefore more closely reflect the natural cellular environment of thetransgene. Such cell lines may be produced by isolating cells (e.g.,fibroblasts or astroglial cells) from a transgenic or homologousrecombinant animal (e.g., a mouse) and culturing them using standardmethods. In some embodiments primary (i.e., non-immortalized) cells arepreferred, or the cells may be may be immortalized (e.g., by theaddition of a gene such as SV40 large T antigen) in order to propagatethem indefinitely in culture.

As used interchangeably herein the terms ‘standard glutamate assay’ or‘standard glutamate transport assay’ (or like terms) are meant toinclude one or more of the following steps:

-   -   a) introducing a recombinant expression vector comprising the        EAAT2 cDNA into a suitable host cells such as COS-7 cells,    -   b) adding detectably-labeled glutamate; and    -   c) detecting glutamate transport in the cells.

Typically, the standard glutamate assay is a sodium-dependent glutamatetransport assay. Introduction of the recombinant vectors in accord withthe standard glutamate assay can be conducted by any acceptable means,e.g., retroviral transfer, viral or bacteriophage infection, calcium-,liposome-, DEAE or polybrene-mediated transfection, biolistic transfer,or other techniques known in the art. See Sambrook, et al. supra;Ausubel, et al. supra.

In one embodiment of the standard glutamate essay, the test and controlcells are washed following introduction of the recombinant vector andthen incubated with a suitable amount of detectably-labeled glutamate,e.g., ³H-labeled glutamate (DuPont-NEN) and non-labeled glutamate.Following a suitable incubation interval, test and control cells arewashed several times in a suitable wash buffer such as ice-cold PBS,solublized in a solution comprising about 0.1% SDS and the amount ofradioactivity associated with the cells determined using conventionalscintillation counting methods.

An especially preferred glutamate transport assay has been disclosed byRothstein et al. (1995) Ann. Neurol. 38: 78. See also Rothstein et al.(1992) N. Engl. J. Med. 326: 1464. The disclosures of which arespecifically incorporated by reference.

III. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., nucleic acids, peptides, peptidomimetics, small molecules,or other drugs) which bind to the EAAT2 promoter, and/or which have astimulatory or inhibitory effect on, for example, EAAT2 promoteractivity.

In one embodiment, the invention provides assays for screening candidateor test compounds which are modulators EAAT2 promoter activity. Inanother embodiment, the invention provides assays for screeningcandidate or test compounds which bind to or modulate the activity of anEAAT2 promoter. The test compounds of the present invention can beobtained using any of the numerous approaches in combinatorial librarymethods known in the art, including: biological libraries; spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the ‘one-bead one-compound’library method; and synthetic library methods using affinitychromatography selection. The biological library approach is limited topeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam, K. S. (1997) Anticancer Drug Des. 12:45).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example, in: DeWitt et al. (1993) Proc. Natl.Acad. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten (992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In a preferred embodiment, an assay is a cell-based assay in which acell which expresses a reporter gene operatively linked to an EAAT2promoter or portion thereof (e.g., whose expression is under the controlof the EAAT2 promoter or portion thereof) is contacted with a testcompound and the ability of the test compound to modulate EAAT-2promoter activity is determined. Determining the ability of the testcompound to modulate EAAT2 promoter activity can be accomplished bymonitoring reporter gene expression (e.g., reporter mRNA or polypeptideexpression level) or activity, for example. As described elsewhereherein, the reporter can be any detectable marker. For example, thereporter can be a nucleic acid sequence, the expression of which can bemeasured by, for example, Northern blotting, RT-PCR, primer extension,or nuclease protection assays. The reporter may also be a nucleic acidsequence that encodes a polypeptide, the expression of which can bemeasured by, for example, Western blotting, ELISA, or RIA assays.Reporter expression can also be monitored by measuring the activity ofthe polypeptide encoded by the reporter using, for example, a standardglutamate transport assay, a luciferase assay, a β-galactosidase assay,a chloramphenicol acetyl transferase (CAT) assay, or a fluorescentprotein assay.

The level of expression or activity of a reporter under the control ofthe EAAT2 promoter in the presence of the candidate compound is comparedto the level of expression or activity of the reporter in the absence ofthe candidate compound. The candidate compound can then be identified asa modulator of EAAT2 promoter activity based on this comparison. Forexample, when expression of reporter mRNA or protein expression oractivity is greater (statistically significantly greater) in thepresence of the candidate compound than in its absenice, the candidatecompound is identified as a stimulator of EAAT2 promoter activity.Alternatively, when expression or activity of reporter mRNA or proteinis less (statistically significantly less) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as an inhibitor of EAAT2 promoter activity.

The ability of the test compound to bind to the EAAT2 promoter and/or tomodulate the binding of proteins (e.g., transcription factors) to theEAAT2 promoter can also be determined. Determining the ability of thetest compound to bind to and/or modulate EAAT2 promoter binding to abinding protein can be accomplished, for example, by coupling the testcompound, the EAAT2 promoter or the binding protein with a radioisotopeor enzymatic label such that binding of the EAAT2 promoter to the testcompound or the binding protein can be determined by detecting thelabeled component in a complex. For example, compounds (e.g., the testcompound, the EAAT2 promoter, or a binding protein) can be labeled with³²p, 125I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and theradioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, compounds can be enzymaticallylabeled with, for example, horseradish peroxidase, alkaline phosphatase,or luciferase, and the enzymatic label detected by determination ofconversion of an appropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound (e.g., a test compound or EAAT2 promoter binding protein)to interact with the EAAT2 promoter without the labeling of any of theinteractants. For example, a microphysiometer can be used to detect theinteraction of a compound with the EAAT2 promoter without the labelingof either the compound or the EAAT2 promoter (McConnell, H. M. et al.(1992) Science 257:1906-1912). As used herein, a “microphysiometer”(e.g., Cytosensor) is an analytical instrument that measures the rate atwhich a cell acidifies its environment using a light-addressablepotentiometric sensor (LAPS). Changes in this acidification rate can beused as an indicator of the interaction between a compound and the EAAT2promoter.

In another embodiment, the assay is a cell-free assay in which an EAAT2promoter or portion thereof is contacted with a test compound and theability of the test compound to modulate (e.g., stimulate or inhibit)the activity of the EAAT2 promoter or portion thereof is determined.Determining the ability of the test compound to modulate the activity ofan EAAT2 promoter can be accomplished, for example, by determining theability of the EAAT2 promoter to bind to an EAAT2 promoter targetmolecule by one of the methods described above for determining directbinding. Determining the ability of the EAAT2 promoter to bind to anEAAT2 promoter target molecule can also be accomplished using atechnology such as real-time Biomolecular Interaction Analysis (BIA).Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 andSzabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein,“BIA” is a technology for studying biospecific interactions in realtime, without labeling any of the interactants (e.g., BIAcore). Changesin the optical phenomenon of surface plasmon resonance (SPR) can be usedas an indication of real-time reactions between biological molecules.

In yet another embodiment, the cell-free assay involves contacting anEAAT2 promoter or portion thereof with a known compound which binds theEAAT2 promoter (e.g., a component of the basal transcription machinery)to form an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith the EAAT2 promoter, wherein determining the ability of the testcompound to interact with the EAAT2 promoter comprises determining theability of the EAAT2 promoter to preferentially bind to or modulate theactivity of an EAAT2 promoter target molecule.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either EAAT2 promoter orits target molecule to facilitate separation of complexed fromuncomplexed forms of one or both of the molecules, as well as toaccommodate automation of the assay. Binding of a test compound to anEAAT2 promoter, or interaction of an EAAT2 promoter with a substrate ortarget molecule in the presence and absence of a candidate compound, canbe accomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase/targetfusion proteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized micrometer plates,which are then combined with the test compound or the test compound andeither the non-adsorbed target protein or EAAT2 promoter, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads or microtiter plate wells are washed to remove any unboundcomponents, the matrix immobilized in the case of beads, complexdetermined either directly or indirectly, for example, as describedabove. Alternatively, the complexes can be dissociated from the matrix,and the level of EAAT2 promoter binding or activity determined usingstandard techniques.

Other techniques for immobilizing proteins or nucleic acids on matricescan also be used in the screening assays of the invention. For example,either an EAAT2 promoter or an EAAT2 promoter substrate or targetmolecule can be immobilized utilizing conjugation of biotin andstreptavidin. Biotinylated EAAT2 promoter, substrates, or targetmolecules can be prepared from biotin-NHS (N-hydroxy-succinimide) usingtechniques known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Alternatively, antibodies reactive withEAAT2 promoter or target molecules but which do not interfere withbinding of the EAAT2 promoter to its target molecule can be derivatizedto the wells of the plate, and unbound target or EAAT2 promoter trappedin the wells by antibody conjugation. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the EAAT2 promoter or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the EAAT2 promoter or target molecule.

In yet another aspect of the invention, the EAAT2 promoter can be usedas “bait” in a one-hybrid assay (see, e.g., BD Matchmaker One-HybridSystem (1995) Clontechniques X(3):2-4; BD Matchmaker LibraryConstruction & Screening Kit (2000) Clontechniques XV(4):5-7; BD SMARTtechnology overview (2002) Clontechniques XVII(1):22-28; Ausubel, F. M.,et al. (1998 et seq.) Current Protocols in Molecular Biology Eds.Ausubel, F. M., et al., pp. 13.4.1-13.4.10) to identify proteins whichbind to or interact with the EAAT2 promoter (“EAAT2 promoter-bindingproteins” or “EAAT2 promoter-bp”) and are involved in EAAT2 promoteractivity. Such EAAT2 promoter-binding proteins are also likely to beinvolved in the regulation of transcription from the EAAT2 promoter.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell-free assay, and the abilityof the agent to modulate the activity of an EAAT2 promoter can beconfirmed in vivo, e.g., in an animal such as an animal model for aneurological disease.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model (e.g., an animal model for a neurologicaldisease). For example, an agent identified as described herein (e.g., anEAAT2 promoter modulating agent or an EAAT2 promoter binding protein)can be used in an animal model to determine the efficacy, toxicity, orside effects of treatment with such an agent. Alternatively, an agentidentified as described herein can be used in an animal model todetermine the mechanism of action of such an agent. Furthermore, thisinvention pertains to uses of novel agents identified by theabove-described screening assays for treatments as described herein.

IV. Methods of Treatment

In one embodiment, the present invention provides methods of treatingneurological and psychiatric disorders which comprise administering atherapeutically effective amount of a pharmaceutical compositioncomprising an EAAT2 promoter modulator a subject (e.g., a mammal such asa human).

To modulate EAAT2 promoter activity, and thereby modulate EAAT2 geneexpression, e.g., a compound disclosed herein or identified by thescreening assays of the invention, can be administered to a cell or asubject. Administration of an EAAT2 promoter modulator to mammaliancells (including human cells) can modulate (e.g., up- or down-regulateEAAT2 mRNA and/or polypeptide expression, thereby up- or down-regulatingglutamate transport into the cell. In such methods, the EAAT2 promotercan be administered to a mammal (including a human) by known procedures.

The preferred therapeutic methods of the invention (which includeprophylactic treatment) in general comprise administration of atherapeutically effective amount of an EAAT2 promoter modulator to ananimal in need thereof, including a mammal, particularly a human. Suchtreatment will be suitably administered to subjects, particularlyhumans, suffering from, having, susceptible to, or at risk for aneurological or psychiatric disorder. The EAAT2 promoter modulators ofthe invention may be also used in the treatment of any other disordersin which EAAT2 may be implicated.

For therapeutic applications, EAAT2 modulators of the invention may besuitably administered to a subject such as a mammal, particularly ahuman, alone or as part of a pharmaceutical composition, comprising theEAAT2 modulator together with one or more acceptable carriers thereofand optionally other therapeutic ingredients. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

The pharmaceutical compositions of the invention include those suitablefor oral, rectal, nasal, topical (including buccal and sublingual),vaginal or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration. The formulations mayconveniently be presented in unit dosage form, e.g., tablets andsustained release capsules, and in liposomes, and may be prepared by anymethods well know in the art of pharmacy. See, for example, Remington'sPharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa.(17th ed. 1985).

Such preparative methods include the step of bringing into associationwith the molecule to be administered ingredients such as the carrierwhich constitutes one or more accessory ingredients. In general, thecompositions are prepared by uniformly and intimately bringing intoassociation the active ingredients with liquid carriers, liposomes orfinely divided solid carriers or both, and then if necessary shaping theproduct.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, sachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion, or packed in liposomes and as a bolus,etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Molded tablets may be made by molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets optionally may be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

Compositions suitable for topical administration include lozengescomprising the ingredients in a flavored basis, usually sucrose andacacia or tragacanth; and pastilles comprising the active ingredient inan inert basis such as gelatin and glycerin, or sucrose and acacia.

Compositions suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tablets.

Application of the subject therapeutics often will be local, so as to beadministered at the site of interest. Various techniques can be used forproviding the subject compositions at the site of interest, such asinjection, use of catheters, trocars, projectiles, pluronic gel, stents,sustained drug release polymers or other device which provides forinternal access. Where an organ or tissue is accessible because ofremoval from the patient, such organ or tissue may be bathed in a mediumcontaining the subject compositions, the subject compositions may bepainted onto the organ, or may be applied in any convenient way.

It will be appreciated that actual preferred amounts of a given EAAT2modulator of the invention used in a given therapy will vary to theparticular active compound being utilized, the particular compositionsformulated, the mode of application, the particular site ofadministration, the patient's weight, general health, sex, etc., theparticular indication being treated, etc. and other such factors thatare recognized by those skilled in the art including the attendantphysician or veterinarian. Optimal administration rates for a givenprotocol of administration can be readily determined by those skilled inthe art using conventional dosage determination tests.

V. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining EAAT2 promoteractivity, in the context of a biological sample (e.g., a sample ofastroglial cells) to thereby determine whether an individual isafflicted with a disease or disorder, or is at risk of developing adisorder, associated with aberrant or unwanted EAAT2 expression oractivity. The invention also provides for prognostic (or predictive)assays for determining whether an individual is at risk of developing adisorder associated with EAAT2 protein, nucleic acid expression, oractivity. For example, mutations in the EAAT2 promoter can be assayed ina biological sample. Such assays can be used for prognostic orpredictive purpose to thereby prophylactically treat an individual priorto the onset of a disorder characterized by or associated with EAAT2protein, nucleic acid expression or activity.

Another aspect of the invention pertains to monitoring the influence ofagents (e.g., drugs, compounds) on the expression or activity of EAAT2in clinical trials.

These and other agents are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of the EAAT2promoter nucleic acid in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting EAAT2 promoternucleic acid (e.g., genomic DNA) such that the presence of EAAT2promoter nucleic acid is detected in the biological sample. A preferredagent for detecting EAAT2 promoter genomic DNA is a labeled nucleic acidprobe capable of hybridizing to EAAT2 promoter genomic DNA. The nucleicacid probe can be, for example, a EAAT2 promoter nucleic acid of SEQ IDNO:1, 2, 3, or 4, or a portion thereof, such as an oligonucleotide of atleast 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficientto specifically hybridize under stringent conditions to EAAT2 promotergenomic DNA. Other suitable probes for use in the diagnostic assays ofthe invention are described herein.

The present invention also provides diagnostic assays for identifyingthe presence or absence of a genetic alteration characterized byaberrant modification or mutation of an EAAT2 promoter.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting EAAT2 promoter genomicDNA, such that the presence of EAAT2 promoter genomic DNA is detected inthe biological sample, and comparing the presence of EAAT2 promotergenomic DNA in the control sample with the presence of EAAT2 promotergenomic DNA in the test sample.

The invention also encompasses kits for detecting she presence of theEAAT2 promoter in a biological sample. For example, the kit can comprisea labeled compound or agent capable of detecting EAAT2 promoter DNA in abiological sample; means for determining the amount and/or sequence ofthe EAAT2 promoter in the sample; and means for comparing the amountand/or sequence of the EAAT2 promoter in the sample with a standard. Thecompound or agent can be packaged in a suitable container. The kit canfurther comprise instructions for using the kit to detect EAAT2 promoternucleic acid.

2. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant or unwanted EAAT2 promoter activity. As usedherein, the term “aberrant” includes a EAAT2 expression or activitywhich deviates from the wild type EAAT2 promoter activity. Aberrantactivity includes increased or decreased activity, as well as activitywhich does not follow the wild type developmental pattern of expressionor the subcellular pattern of expression. For example, aberrant EAAT2promoter activity is intended to include the cases in which a mutationin the EAAT2 promoter causes the EAAT2 gene to be under-expressed orover-expressed and situations in which such mutations result in anon-functional EAAT2 protein or a protein which does not function in awild-type fashion, e.g., a protein which does not interact with ortransport a EAAT2 substrate (i.e., glutamate), or one which interactswith or transports a non-EAAT2 substrate. As used herein, the term“unwanted” includes an unwanted phenomenon involved in a biologicalresponse such as deregulated glutamate transport. For example, the termunwanted includes a EAAT2 expression or activity which is undesirable ina subject.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in EAAT2promoter activity, such as neurological or psychiatric disorder.Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing a disorder associated with amisregulation in EAAT2 promoter activity, such as a neurological orpsychiatric disorder. Thus, the present invention provides a method foridentifying a disease or disorder associated with aberrant or unwantedEAAT2 promoter activity in which a test sample is obtained from asubject and EAAT2 promoter nucleic acid (e.g., genomic DNA) is detected(and/or sequenced), wherein the presence of an EAAT2 promotermodification or mutation is diagnostic for a subject having or at riskof developing a disease or disorder associated with aberrant or unwantedEAAT2 promoter activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid, cell sample, or tissue, and ispreferably astroglial cells.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted EAAT2 promoter activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a neurological or psychiatricdisorder. Thus, the present invention provides methods for determiningwhether a subject can be effectively treated with an agent for adisorder associated with aberrant or unwanted EAAT2 promoter activity inwhich a test sample is obtained and EAAT2 promoter activity is detected.

The methods of the invention can also be used to detect geneticalterations in a EAAT2 promoter, thereby determining if a subject withthe altered gene is at risk for a disorder characterized bymisregulation in EAAT2 protein activity or nucleic acid expression, suchas a neurological or psychiatric disorder. In preferred embodiments, themethods include detecting, in a sample of cells from the subject, thepresence or absence of a genetic alteration characterized by at leastone of an alteration affecting the integrity of the EAAT2 promoter. Forexample, such genetic alterations can be detected by ascertaining theexistence of at least one of 1) a deletion of one or more nucleotidesfrom an EAAT2 promoter; 2) an addition of one or more nucleotides to aEAAT2 promoter; 3) a substitution of one or more nucleotides of an EAAT2promoter, 4) a chromosomal rearrangement of an EAAT2 promoter; 5)aberrant modification of a EAAT2 promoter, such as of the methylationpattern of the genomic DNA, and 6) allelic loss of an EAAT2 promoter. Asdescribed herein, there are a large number of assays known in the artwhich can be used for detecting alterations in an EAAT2 promoter. Apreferred biological sample is a tissue or serum sample isolated byconventional means from a subject.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the EAAT2 promoter(see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This methodcan include the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic DNA) from the cells of the sample,contacting the nucleic acid sample with one or more primers whichspecifically hybridize to a EAAT2 promoter under conditions such thathybridization and amplification of the EAAT2 promoter (if present)occurs, and detecting the presence or absence of an amplificationproduct, or detecting the size of the amplification product andcomparing the length to a control sample. It is anticipated that PCRand/or LCR may be desirable to use as a preliminary amplification stepin conjunction with any of the techniques used for detecting mutationsdescribed herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in the EAAT2 promoter can beidentified by hybridizing a sample and control nucleic acids, e.g., DNAor RNA, to high density arrays containing hundreds or thousands ofoligonucleotides probes (Cronin, M. T. et al. (1 996) Hum. Mutat.7:244-255; Kozal, M. J. et al. (1 996) Nat. Med. 2:753-759). Forexample, genetic mutations in the EAAT2 promoter can be identified intwo dimensional arrays containing light-generated DNA probes asdescribed in Cronin et al. (1996) supra. Briefly, a first hybridizationarray of probes can be used to scan through long stretches of DNA in asample and control to identify base changes between the sequences bymaking linear arrays of sequential overlapping probes. This step allowsthe identification of point mutations. This step is followed by a secondhybridization array that allows the characterization of specificmutations by using smaller, specialized probe arrays complementary toall variants or mutations detected. Each mutation array is composed ofparallel probe sets, one complementary to the wild-type gene and theother complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the EAAT2 promoter anddetect mutations by comparing the sequence of the sample EAAT2 promoterwith the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplatedthat any of a variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W. (1995) Biotechniques19:448), including sequencing by mass spectrometry (see, e.g., PCTInternational Publication No. WO 94/16101; Cohen et al. (1996) Adv.Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem.Biotechnol. 38:147-159).

Other methods for detecting mutations in the EAAT2 promoter includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al.(1985) Science 230:1242). In general, the art technique of “mismatchcleavage” starts by providing heteroduplexes formed by hybridizing(labeled) RNA or DNA containing the wild-type EAAT2 promoter sequencewith potentially mutant RNA or DNA obtained from a tissue sample. Thedouble-stranded duplexes are treated with an agent which cleavessingle-stranded regions of the duplex such as which will exist due tobasepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digesting the mismatchedregions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can betreated with hydroxylamine or osmium tetroxide and with piperidine inorder to digest mismatched regions. After digestion of the mismatchedregions, the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, for example,Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al.(1992) Methods Enzymol. 217:286-295. In a preferred embodiment, thecontrol DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in EAAT2 cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a EAAT2 promotersequence, e.g., a wild-type EAAT2 promoter sequence, is hybridized to acDNA or other DNA product from a test cell(s). The duplex is treatedwith a DNA mismatch repair enzyme, and the cleavage products, if any,can be detected from electrophoresis protocols or the like. See, forexample, U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in the EAAT2 promoter. For example, singlestrand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766,see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992)Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments ofsample and control EAAT2 promoter nucleic acids will be denatured andallowed to renature. The secondary structure of single-stranded nucleicacids varies according to sequence, the resulting alteration inelectrophoretic mobility enables the detection of even a single basechange. The DNA fragments may be labeled or detected with labeledprobes. The sensitivity of the assay may be enhanced by using RNA(rather than DNA), in which the secondary structure is more sensitive toa change in sequence. In a preferred embodiment, the subject methodutilizes heteroduplex analysis to separate double stranded heteroduplexmolecules on the basis of changes in electrophoretic mobility (Keen etal. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving an EAAT2 promoter.

Furthermore, any cell type or tissue in which EAAT2 is expressed (e.g.,astroglial cells) may be utilized in the prognostic assays describedherein.

3. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs) on the activity of theEAAT2 promoter can be applied not only in basic drug screening, but alsoin clinical trials. For example, the effectiveness of an agentdetermined by a screening assay as described herein to increase EAAT2promoter activity, can be monitored in clinical trials of subjectsexhibiting decreased EAAT2 promoter activity. Alternatively, theeffectiveness of an agent determined by a screening assay to decreaseEAAT2 promoter activity, can be monitored in clinical trials of subjectsexhibiting increased EAAT2 promoter activity. In such clinical trials,the expression or activity of a EAAT2 promoter, and preferably, othergenes that have been implicated in, for example, a neurological orpsychiatric disorder can be used as a “read out” or markers of thephenotype of a particular cell.

For example, and not by way of limitation, genes, including EAAT2, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates EAAT2 promoter activity (e.g.,identified in a screening assay as described herein) can be identified.Thus, to study the effect of agents on neurological or psychiatricdisorders, for example, in a clinical trial, cells can be isolated andRNA prepared and analyzed for the levels of expression of EAAT2 andother genes implicated in the disorder, respectively. The levels of geneexpression (e.g., a gene expression pattern) can be quantified bynorthern blot analysis or RT-PCR, as described herein, or alternativelyby measuring the amount of protein produced, by one of the methods asdescribed herein, or by measuring the levels of activity of EAAT2 orother genes. In this way, the gene expression pattern can serve as amarker, indicative of the physiological response of the cells to theagent. Accordingly, this response state may be determined before, and atvarious points during treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) including the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a EAAT2 protein,mRNA, or genomic DNA in the preadministration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of expression or activity of the EAAT2 protein, mRNA, orgenomic DNA in the post-administration samples; (v) comparing the levelof expression or activity of the EAAT2 protein, mRNA, or genomic DNA inthe pre-administration sample with the EAAT2 protein, mRNA, or genomicDNA in the post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of EAAT2 to higher levels than detected, i.e., toincrease the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of EAAT2 to lower levels than detected, i.e., to decrease theeffectiveness of the agent. According to such an embodiment, EAAT2expression or activity may be used as an indicator of the effectivenessof an agent, even in the absence of an observable phenotypic response.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the sequence listing and the figures, areincorporated herein by reference.

EXAMPLES Example 1 Identification and Cloning of the Eaat2 Promoter

A genomic DNA PAC clone known from a BLAST search to contain the 5′ endof the EAAT2 gene was obtained from the Sanger Institute, Cambridge,United Kingdom. The clone was grown up according to the supplier'sinstructions, and DNA was isolated and digested with EcoRI. Fragmentswere resolved on a 0.8% agarose gel and transferred to a nitrocellulosemembrane. The blot was probed with a random-primed 130 bp PCR productcontaining EAAT2 exon 1. An 8 kb EcoRI fragment generated a positivehybridization signal, and this fragment was subsequently subcloned intoa TA-vector. Sequence data revealed that this fragment did contain exon1 and additional sequence upstream of exon 1 (2.8 kb of the 8 kbfragment), which was obtained and analyzed for promoter motifs.

Example 2 Analysis of the EAAT2 Promoter

The sequence of the EAAT2 promoter and flanking DNA is shown in FIGS.1A-1B and set forth as SEQ ID NO:1. This sequence was analyzed using thePROSCAN Version 1.7 suite of software programs developed by Dr. DanPrestridge (Prestridge, D. S. (1995) J. Mol. Biol. 249: 923-32), whichare designed to find putative eukaryotic Pol II promoter sequences inprimary sequence data. Potential promoter elements identified using thisanalysis include CCAAT boxes, SpI binding sites (GGGGCGGGG orCCCCGCCCC), E-box motifs (CACCTG, CAYGTG or CANNTG), binding sites forelements from the GATA family of transcription factors (motifs that canaffect kidney tissue expression), NF-κB, and binding sites for WT1(GNGGGNGNG). Nucleotide repeat regions, poly(dA:dT) and poly(dG:dT),that are thought to affect transcription through conformational changesin the DNA structure were also found in the flanking sequence. Notably,cyclic AMP response elements (CREB binding domains) were also identifiedin this first 2.8 kb fragment. As described elsewhere herein,GLT-1/EAAT2 is known to be upregulated by cyclic AMP, which typicallyactivates transcription thru cyclic AMP response elements (CREB).Several possible CREB promoter motifs were also found within the EAAT2promoter. A schematic representation of the EAAT2 promoter regionshowing the identified promoter elements is shown in FIG. 2. The fullresults of the PROSCAN analysis are set forth below in Tables I-VI.

TABLE I Promoter region predicted on forward strand in 2059 to 2309Promoter Score: 60.44 (Promoter Cutoff = 53.000000) Significant Signals:Name TFD # Strand Location Weight GCF S01964 + 2126 2.361000 AP-2S00346 + 2129 1.355000 Sp1 S00802 + 2129 3.292000 GCF S01964 − 21322.284000 AP-2 S01936 + 2132 1.108000 Sp1 S00978 − 2134 3.361000 Sp1S00333 − 2135 3.442000 (Sp1) S00857 − 2136 4.876000 JCV_repeated_sequencS01193 + 2171 1.427000 Sp1 S00801 + 2187 2.755000 Sp1 S00781 − 21922.772000 UCE.2 S00437 − 2207 1.216000 UCE.2 S00437 + 2241 1.278000 UCE.2S00437 − 2285 1.216000 AP-2 S01936 + 2295 1.108000 EARLY-SEQ1 S01081 +2295 6.322000 (Sp1) S01187 + 2295 8.117000 Sp1 S00801 + 2296 2.755000AP-2 S00346 + 2297 1.355000 Sp1 S00802 + 2297 3.292000 Sp1 S00781 − 23012.772000 SP1 S00978 − 2302 3.361000 JCV_repeated-sequenc S01193 − 23021.658000

TABLE II Promoter region predicted on forward strand in 3187 to 3437Promoter Score: 54.08 (Promoter Cutoff = 53.000000) Significant Signals:Name TFD # Strand Location Weight Sp1 S00801 + 3187 2.755000 AP-2S00346 + 3188 1.355000 Sp1 S00781 − 3192 2.772000 AP-2 S01936 + 32021.108000 AP-2 S00180 + 3204 1.863000 Sp1 S00978 + 3208 3.013000 Sp1S00977 + 3208 7.086000 JCV_repeated_sequenc S01193 + 3208 1.427000 AP-2S00346 − 3213 1.672000 Sp1 S00802 − 3213 3.061000 EARLY-SEQ1 S01081 −3215 5.795000 AP-2 S01936 − 3218 1.091000 GCF S01964 + 3229 2.361000 Sp1S00781 + 3312 3.191000 JCV_repeated_sequenc S01193 + 3315 1.427000 Sp1S00801 − 3317 3.119000 Sp1 S00978 + 3323 3.013000 Sp1 S00802 − 33283.061000 UCE.2 S00437 + 3357 1.278000 UCE.2 S00437 − 3360 1.216000 AP-2S01936 + 3375 1.108000 T-Ag S00974 + 3430 1.086000 (Sp1) S01027 − 34362.233000

TABLE III Promoter region predicted on forward strand in 3820 to 4070Promoter Score: 94.21 (Promoter Cutoff = 53.000000) Significant Signals:Name TFD # Strand Location Weight GCF S01964 + 3820 2.361000TTR_inverted_repeat S01112 − 3827 3.442000 AP-2 S01936 + 3828 1.108000AP-2 S00180 + 3828 1.863000 GCF S01964 − 3828 2.284000 Sp1 S00781 + 38403.191000 Sp1 S00801 − 3845 3.119000 T-Ag S00974 + 3893 1.086000 Sp1S00979 + 3893 6.023000 Sp1 S00645 + 3893 12.906000 Sp1 S00064 + 389310.681000 Sp1 S01542 + 3893 6.661000 JCV_repeated_sequenc S01193 + 38941.427000 Sp1 S00978 + 3894 3.013000 AP-2 S01936 − 3895 1.091000 GCFS01964 + 3896 2.361000 Sp1 S00802 − 3899 3.061000 EARLY-SEQ1 S01081 −3901 5.795000 (Sp1) S01187 − 3901 6.819000 APRT-mouse_US S00216 − 39027.604000 UCE.2 S00437 − 3922 1.216000 GCF S01964 − 3967 2.284000 NF-kBS01644 − 4004 50.000000 JCV_repeated_sequenc S01193 + 4022 1.427000 Sp1S00781 + 4052 3.191000 E2F S01247 + 4053 25.816999 E2F S01242 + 405317.211000 Sp1 S00801 − 4057 3.119000 E2F S01952 − 4061 6.454000

TABLE IV Promoter region predicted on reverse strand in 3565 to 3315Promoter Score: 70.71 Promoter Cutoff = 53.000000) TATA found at 3350,Est.TSS = 3318 Significant Signals: Name Strand Location Weight Sp1 −3561 2.755000 GCF + 3557 2.284000 Sp1 + 3556 2.772000 T-Ag − 35251.086000 c-fos.5 − 3522 1.912000 AP-2 − 3513 1.108000 MLTF − 35061.157000 AP-2 + 3375 1.091000 UCE.2 − 3360 1.278000 UCE.2 + 33571.216000 Sp1 − 3329 2.755000 Sp1 − 3328 3.292000 Sp1 + 3324 2.772000Sp1 + 3323 3.361000 JCV_repeated_sequenc + 3315 1.658000

TABLE V Promoter region predicted-on reverse strand in 3065 to 2815Promoter Score: 77.12 (Promoter Cutoff = 53.000000) Significant Signals:Name Strand Location Weight TTR_inverted_repeat + 3055 3.442000 Sp1 −3051 3.191000 Sp1 + 3046 3.119000 GCF − 2991 2.361000 AP-2 + 29811.091000 AP-2 + 2980 1.064000 AP-2 + 2979 1.672000 AP-2 + 2979 1.721000UCE.2 − 2969 1.278000 UCE.2 + 2941 1.216000 JCV_repeated_sequenc − 29391.427000 APRT-mouse US − 2919 6.003000 AP-2 − 2918 1.108000 Sp1 − 29172.755000 GCF + 2913 2.284000 Sp1 + 2912 2.772000 GCF − 2854 2.361000 Sp1− 2823 3.292000 AP-2 − 2823 1.355000 AP-2 − 2820 1.108000 S Sp1 − 28199.386000 EARLY-SEQ1 − 2818 6.322000 (Sp1) − 2818 8.117000 Sp1 + 28183.361000 Sp1 − 2817 2.755000 (Sp1) + 2816 4.876000

TABLE VI Promoter region predicted on reverse strand in 2445 to 2195Promoter Score: 53.54 (Promoter Cutoff = 53.000000) Significant Signals:Name Strand Location Weight GCF − 2401 2.361000 UCE.2 + 2396 1.216000AP-2 − 2382 1.355000 AP-2 − 2381 1.108000 Sp1 − 2379 2.755000JCV_repeated_sequenc + 2379 1.658000 H4TF1 + 2377 2.099000 Sp1 + 23742.772000 (Sp1) − 2311 4.589000 T-Ag − 2310 1.086000 Sp1 − 2309 3.013000AP-2 + 2308 1.091000 Sp1 − 2305 3.191000 Sp1 + 2304 3.061000JCV_repeated_sequenc − 2302 1.427000 Sp1 + 2300 3.119000 AP-2 + 22971.672000 (Sp1) + 2295 6.819000 EARLY-SEQ1 + 2295 5.795000 UCE.2 − 22851.278000 UCE.2 + 2241 1.216000 UCE.2 − 2207 1.278000 SDR_RS − 22031.554000 AP-2 + 2198 1.091000

Three regions derived from different restriction enzyme digests of the5′ EAAT2 flanking region of the promoter (P1, P2, P3) were identified:an ˜2.8 kb KpnI-NcoI fragment (P1), an ˜2.5 kb KpnI-SaII fragment (P2)and an ˜0.86 kb SmaI-SaII fragment (P3). The P1, P2, and P3 fragmentswere amplified by PCR and cloned into a promoterless luciferase reportervector (pGL3, Promega, Madison, Wis.). The constructs were transientlytransfected into various cell lines, including HEK 293T cells, COS7cells, and C6 glioma cells, and luciferase activity was measured. Thehighest level of expression was obtained from COS7 cells. To control fortransfection efficiency, luciferase activities were normalized withRenilla luciferase activity or β-galactosidase activity (FIG. 3). Inpreliminary studies, the 2.8 upstream EAAT2 promoter fragment wasoperatively linked with the EAAT2 cDNA. In transient transfection, EAAT2protein was detected in COS-7 cells containing this construct.

Example 3 Production of EAAT2 Transgenic Mice

Two independent lines of transgenic mice based on the EAAT2 promoterwere created. The first line contained the P1 fragment linked toenhanced green fluorescent protein (eGFP). This DNA construct wassubcloned into a StealthGene vector (Tosk, Inc., Santa Cruz, Calif.)that contains a modified P transposable element (see U.S. Pat. No.6,291,243). The vector was injected into adult mice (C57), andapproximately 40% of all cells, including the reproductive cells, haveinserted, active cDNA. The mice were mated with wild-type mice toproduce a colony of “founders”. These mice have been mated and haveconfirmed transgenic expression.

A second line of transgenic mice are created using a BAC clone. The BACtransgenic approach is described in Yang, X. W. et al. (1997) Nat.Biotechnol. 15(9):859-65, incorporated herein by reference. The BACclone shown in FIG. 4 was chosen to include approximately 100 kbupstream of the EAAT2/GLT-1 coding sequence, as well as at least 120 kbthat comprises the estimated EAAT2 gene. With targeted modification of aBAC clone containing a specific gene of interest (e.g., EAAT2), andsubsequent germline transmission in transgenic mice, reporter cassettesmay be used to study expression driven by the gene's promoter withoutspecifically identifying promoter regulatory elements. One specificstrategy that has been developed for manipulation of BAC DNA useshomologous recombination, mediated via the RecA protein, between a RecA+shuttle vector containing the modification cassette and BAC DNA in RecA−host bacteria as described in Yang et al. (1997) supra. An eGFP reportergene will be inserted at the initiation of the coding sequence using theshuttle vector. A schematic showing the protocol for generating the miceis shown in FIG. 5.

The invention has been described in detail with reference to preferredembodiments thereof. However, it will be appreciated that those skilledin the art, upon consideration of this disclosure, may make modificationand improvements within the spirit and scope of the invention as setforth in the following claims.

1. An isolated nucleic acid molecule consisting of the nucleic acid ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or a fragmentthereof, wherein said fragment has EAAT2 promoter activity and comprisesa promoter element selected from the group consisting of: a CAATT box,an SP1 binding site, an E-box motif, a GATA family transcription factorbinding site, an NF-κB binding site, a WT1 binding site, a poly(dG:dT)repeat region, or a CREB binding site.
 2. A vector comprising thenucleic acid molecule of claim
 1. 3. The vector of claim 2, which is anexpression vector.
 4. An isolated host cell transfected with theexpression vector of claim
 3. 5. An isolated nucleic acid moleculecomprising the nucleic acid molecule of claim 1 and a cDNA moleculeoperably linked to said nucleic acid molecule.
 6. The nucleic acidmolecule of claim 5, wherein the cDNA molecule comprises the EAAT2 cDNAsequence.
 7. The nucleic acid molecule of claim 5, wherein the cDNAmolecule comprises a reporter gene.
 8. The nucleic molecule of claim 7,wherein the reporter gene is selected from the group consisting ofluciferase, .beta.-galactosidase, chloramphenicol acetyl transferase,and a fluorescent protein.
 9. The nucleic acid molecule of claim 8,wherein the luciferase is selected from the group consisting of fireflyluciferase and Renilla luciferase.
 10. The nucleic acid molecule ofclaim 8, wherein the fluorescent protein is selected from the groupconsisting of green fluorescent protein, red fluorescent protein, yellowfluorescent protein, blue fluorescent protein, and cyan fluorescentprotein.
 11. A vector comprising the nucleic acid molecule of claim 5.12. The vector of claim 11, which is an expression vector.
 13. Anisolated host cell transtected with the expression vector of claim 12.14. A method of producing an mRNA molecuie or a polypeptide comprisingculturing the host cell of claim 13 in an appropriate culture medium to,thereby, produce the mRNA molecule or the polypeptide, wherein said mRNAmolecule or polypeptide is encoded by the cDNA molecule.
 15. The methodof claim 14, wherein the cDNA molecule comprises the EAAT2 cDNAsequence.
 16. The method of claim 14, wherein the cDNA moleculecomprises a reporter gene.
 17. The method of claim 16, wherein thereporter gene is selected from the group consisting of luciferase,.beta.-galactosidase, chloramphenicol acetyl transferase, and afluorescent protein.
 18. The method of claim 17, wherein the luciferaseis selected from the group consisting of firefly luciferase and Renillaluciferase.